Molecular basis and modulation of target DNA recognition in the type IV restriction endonuclease McrBC

Abstract

Target DNA recognition is a crucial factor that drives the cellular functions carried out by DNA binding proteins. An increasing number of DNA binding proteins that read epigenetic modifications on DNA bases and certain DNA sequences in the genome are found to use a mechanism of DNA base flipping, wherein the target base is rotated out of the DNA helix into a recognition pocket of the enzyme. One feature that is common to these base flipping enzymes is the presence of a protein residue that intercalates into the DNA cavity and fills the space left out by the flipped base. To understand the role of this residue in target DNA recognition and in the base flipping mechanism in DNA reader proteins, a restriction enzyme McrBC which utilizes DNA base flipping for recognizing methylcytosine in DNA, was used as the study system. A series of mutants of this enzyme was studied, which carried amino acids of varying size and hydrophobicity as the DNA intercalating residue. Analysis of the target recognition in these mutants through enzyme activity assays and DNA binding assays indicated that modulation of the base flipping process by specifically varying the size and hydrophobicity of the intercalating residue resulted in generation of enzyme constructs that differ in target recognition and enzymatic efficiencies. These observations indicate that the intercalating residue directly plays a role in mediating target recognition and enzymatic efficiency in DNA reader proteins by affecting base flipping. These insights will have further implications in the field of enzyme engineering and protein design for base flipping proteins.